Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been an important analytical method for a variety of fields, especially related to analysis of synthetic and biopolymers. The method has been extended from a high vacuum (HV) technique in which time-of-flight (TOF) mass analyzers provided nearly unlimited mass range to intermediate pressure (IP) and atmospheric pressure (AP) techniques interfaced to mass analyzers having limited mass-to-charge (m/z) range. Production of singly charged ions on these m/z limited instruments, however, eliminated the ability to mass analyze high-mass compounds. Small molecule analysis is also limited by the chemical background associated with the ionization of the desired analyte e.g., drugs and metabolites, at any of the pressure regimes used. Inducing fragmentation using collision induced dissociation (CID) of singly charged ions produces little sequence information; newer and more potent fragmentation methods such as electron transfer dissociation (ETD) and electron capture dissociation (ECD) are not applicable to singly charged analyte ions. MALDI operates from the solid state and is a surface method enabling surface imaging approaches to determine the localization of certain analytes within a surface. Commercial MALDI ion source technology operates the laser in reflection geometry limiting the spatial resolution and speed of analysis. To increase the speed, expensive high repetition lasers can be employed more rapidly enabling the measurement of ˜100 shots of sub-spectra to be combined as to what is referred to in MALDI as a mass spectrum and in case of imaging of surfaces used to determine analytes location within a surface employing respective computing programs. Advantages of singly charged ions are the simplicity of data interpretation, especially of complex mixtures.
Electrospray ionization (ESI) is an ionization method whereby a voltage, usually several thousand volts, is placed between a capillary through which a solution is passed and a counter electrode which contains the entrance to the vacuum of the mass spectrometer. Highly charged liquid droplets are formed in the ESI process and desolvation of these droplets leads to formation of bare ions that are sampled by the mass spectrometer. While the MALDI method produces primarily singly charged ions, the ESI liquid introduction method produces ions of high charge states if multiple ionization sites exist on the analyte molecule. Small molecules such as drugs and small peptides and lipids produce singly charged ions. Improved characterization is achieved utilizing multiply charged ions using activation methods including but not limited to CID, ETD, and ECD for powerful fragmentation of the analytes at will in the mass analyzer for sequence or structural information including but not limited to posttranslational modifications of analytes including but not limited to peptides and proteins intact and enzymatically digested. The disadvantage is the complexity and data interpretation associated with multiply charged ions especially with increasing complexity of the analyte and that sprayable conditions need to be achieved limiting applications to systems that can be solubilized and “sprayed”.
Inlet ionization methods include laserspray ionization (LSI), matrix assisted inlet ionization (MAII), and solvent assisted inlet ionization (SAII) producing abundant highly charged ions without the use of a voltage from the solid state (MAII, LSI) or solution (SAII). Laserspray ionization (LSI) MS is a surface method that has the potential to characterize macromolecular structures directly from their native and complex environment with high spatial resolution important in surface imaging such as tissue. LSI was introduced on high performance mass spectrometers (Orbitrap, SYNAPT G2, LTQ Velos with ETD capabilities) operating at AP without the application of any electrical field demonstrating its usefulness for tissue analysis and surface imaging of e.g., lipids, peptides, proteins, and synthetic polymers on mass range limited mass spectrometers. The production of highly charged ions directly from surfaces in high abundance allows sequencing of for example peptides and proteins using ETD. Chemical background associated with LSII is minute. While LSI at AP offers many advantages, there is still room for improvement, especially relative to sensitivity limitations associated with the transfer of ions from AP-to-vacuum, and additional systems and methods for use in the analysis of macromolecular structures.